Membrane fusion events are required in three steps in sea urchin fertilization: the acrosome reaction in sperm, fusion of the plasma membrane of acrosome-reacted sperm with the plasma membrane of the egg, and exocytosis of the contents of the egg cortical granules. We recently reported the involvement of a Zn2+-dependent metalloendoprotease in the acrosome reaction (Farach, H. C., D. I. Mundy, W. J. Strittmatter, and W. J. Lennarz. 1987. J. Biol. Chem. 262:5483-5487). In the current study, we investigated the possible involvement of metalloendoproteases in the two other fusion events of fertilization. The use of inhibitors of metalloendoproteases provided evidence that at least one of the fusion events subsequent to the acrosome reaction requires such enzymes. These inhibitors did not block the binding of sperm to egg or the process of cortical granule exocytosis. However, sperm-egg fusion, assayed by the ability of the bound sperm to establish cytoplasmic continuity with the egg, was inhibited by metalloendoprotease substrate. Thus, in addition to the acrosome reaction, an event in the gamete fusion process requires a metalloendoprotease.